Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Genetically engineered bacteria expressing oxalate oxidase recombinantly and its construction method and application

A technology of oxalate oxidase and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of low content, complicated extraction and purification process, and cumbersome procedures

Active Publication Date: 2021-04-06
WUHAN KANGFUDE BIOTECH CO LTD
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although oxalate oxidase exists widely in plants, its content is low and the extraction and purification process is complicated
The expression level of oxalate oxidase in the expression hosts of other microorganisms (Pichia pastoris, Bacillus, etc.) is generally not high, and it can be expressed in the prokaryotic expression system E.coli but insoluble, and the inclusion bodies need to be cleaned, purified, and refolded. cumbersome procedures

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Genetically engineered bacteria expressing oxalate oxidase recombinantly and its construction method and application
  • Genetically engineered bacteria expressing oxalate oxidase recombinantly and its construction method and application
  • Genetically engineered bacteria expressing oxalate oxidase recombinantly and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: artificially design and synthesize the optimized gene of oxalate oxidase

[0043] This embodiment provides an optimized gene of oxalate oxidase, which is used to encode an oxalate oxidase, and the amino acid sequence of the oxalate oxidase is shown in SEQ ID NO.1. Wherein, the signal peptide sequence of the oxalate oxidase is the 1-17 amino acid sequence shown in SEQ ID NO.1, which is derived from Trichoderma reesei Rut-C30 strain; the mature peptide sequence of the oxalate oxidase is SEQ ID NO.1 The shown 18-458 amino acid sequence is derived from Ceriporiopsis subvermispora.

[0044] Furthermore, the nucleotide sequence of the optimized gene of oxalate oxidase provided in this example is shown in SEQ ID NO.2. The process of artificially designing and synthesizing the optimized gene of the oxalate oxidase is as follows:

[0045]The oxalate oxidase gene derived from Cereus paracereus was optimized according to the codon preference of Trichoderma reesei (...

Embodiment 2

[0046] Embodiment 2: Construction of the pyr4 gene deletion mutant strain Rut-C30(pyr4-) of Trichoderma reesei Rut-C30

[0047] 201. Extraction of Genomic DNA from Trichoderma reesei Rut-C30

[0048] Inoculate the fresh spores of Trichoderma reesei Rut-C30 into the liquid medium for overnight culture, collect the mycelium by filtration, wash twice with sterile water, grind the mycelium with liquid nitrogen, take an appropriate amount of mycelium powder into a 1.5ml centrifuge tube, Use the Ezup column type fungal genomic DNA extraction kit (purchased from Shanghai Shenggong) to isolate genomic DNA, the method is as follows: add 200ul of BufferDigestion (digestion buffer) and 2ul of β-mercaptoethanol, then add 20ul of Proteinase K (protease K) solution, shake and mix. Water bath at 56°C for 1 h until the cells were completely lysed. After the water bath, 20 ul of RNase A (ribonuclease A) with a concentration of 10 mg / ml was added, and left at room temperature for 2 to 5 minut...

Embodiment 3

[0075] Embodiment 3: knockout the mus53 gene in Trichoderma reesei Rut-C30 (pyr4-) bacterial strain

[0076] According to public literature reports (Matthias G. Steiger, APPLIED AND ENVIRONMENTALMICROBIOLOGY, Jan. 2011, p.114–121) the mus53 gene (homologous to the human Lig4 gene) is required for the function of non-homologous end-joining (NHEJ), which Functional disruption can bring about 100% homologous recombination efficiency. In this example, the mus53 gene in the Trichoderma reesei Rut-C30 (pyr4-) strain was knocked out, laying the foundation for the subsequent site-specific integration knock-in example.

[0077] 301. Construction of Trichoderma reesei mus53 gene knockout vector pMDT05-mus53KO

[0078] Referring to the Trichoderma reesei mus53 gene (Protein Id: 58509) information provided in the open literature (MatthiasG.Steiger, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan.2011, p.114–121), search for the mus53 gene in the Trichoderma reesei genome database Locus sequ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a genetically engineered bacterium for recombinantly expressing oxalate oxidase, its construction method and application, and relates to the field of genetic engineering. The method for constructing the genetically engineered bacteria comprises the following steps: optimizing the oxalate oxidase gene derived from Cerexinus worms according to the codon preference of Trichoderma reesei to obtain the optimized gene of oxalate oxidase; knocking out Trichoderma reesei The pyr4 gene and mus53 gene in the genome were used to construct a mutant strain of Trichoderma reesei with simultaneous deletion of the pyr4 gene and mus53 gene; the optimized gene of oxalate oxidase was used to construct a recombinant expression vector; the vector for recombinant expression of oxalate oxidase was used to transform T. reesei A mutant strain of the mold pyr4 gene and mus53 gene deletion at the same time is obtained to obtain a genetically engineered bacterium expressing oxalate oxidase recombinantly. The genetically engineered bacterium of the invention can efficiently express the oxalate oxidase with natural activity, and is suitable for the research and development of recombinant oxalate oxidase medicines and the production of low-oxalate foods and beverages.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a genetically engineered bacterium for recombinantly expressing oxalate oxidase and its construction method and application. Background technique [0002] Oxalic acid (oxalic acid), that is, oxalic acid, the molecular formula is H 2 C 2 o 4 , widely exists in nature, often in the form of oxalate in the cell membranes of plants such as barberry, sheep's foot, sorrel and sorrel, and almost all plants contain oxalate. It is high in certain foods (such as spinach, green tea, coffee, etc.). Oxalic acid is the final product in the body and cannot be further decomposed. The main way of excretion is to excrete it with urine. Oxalic acid in the human body includes exogenous oxalic acid and endogenous oxalic acid. Exogenous oxalic acid refers to oxalic acid ingested through diet. Endogenous oxalic acid refers to oxalic acid produced by the body's own metabolism. Exogenous oxalic acid...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/80C12N1/15A61K38/44A61P13/04C12R1/885
CPCA61K38/00A61P13/04C12N9/0008C12N15/80C12N2800/22C12Y102/03004
Inventor 汪卫汪小锋刘艳红黄荷
Owner WUHAN KANGFUDE BIOTECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com